Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important role in human health. Because animals are unable to synthesize carotenoid de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved source for carotenoids. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives, and colorants in cosmetics, to mention a few.
Industrially, only a few carotenoids are used for food colors, animal feeds, pharmaceuticals, and cosmetics, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol., 70:181-191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis; but, only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. As a result, carotenoids produced from these plants are very expensive. One way to increase the productive capacity of biosynthesis would be to apply recombinant DNA technology (reviewed in Misawa and Shimada, J. Biotech., 59:169-181 (1998)). Thus, it would be desirable to produce carotenoids in non-carotenogenic bacteria and yeasts, thereby permitting control over quality, quantity, and selection of the most suitable and efficient producer organisms. The latter is especially important for commercial production economics (and therefore availability) to consumers.
Carotenoid ketolases are a class of enzymes that introduce keto groups to the ionone ring of the cyclic carotenoids, such as β-carotene, to produce ketocarotenoids. Examples of ketocarotenoids include astaxanthin, canthaxanthin, adonixanthin, adonirubin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, 4-keto-gamma-carotene, 4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene, deoxyflexixanthin, and myxobactone. Two classes of ketolase, CrtW and CrtO, have been reported. The two classes have similar functionality yet appear to have arisen independently as they share very little sequence similarity. The CrtW is a symmetrically acting enzyme that adds keto-groups to both rings of β-carotene (Hannibal et al., J. Bacteriol., 182: 3850-3853 (2000)). Fernández-González et al. (J. of Biol. Chem., 272: 9728-9733 (1997)) reported that the CrtO ketolase enzyme from Synechocystis sp. PCC6803 adds a keto-group asymmetrically to only one of the two β-ionone rings of β-carotene.
Several examples of CrtW ketolases have been reported in variety of bacteria including Agrobacterium aurantiacum (U.S. Pat. No. 6,150,130), Bradyrhizobium sp. (U.S. Patent Publication No. 20030087337), and Brevundimonas aurantiacum (WO 02/079395). However, there is a need to identify additional novel CrtW ketolase genes useful for genetically engineering industrially suitable microorganisms for the production of valuable ketocarotenoids, such as canthaxanthin and astaxanthin. Additionally, there is a particularly important need to identify CrtW type ketolases having relatively low to moderate sequence homology (i.e. <65% nucleotide sequence identity) as coexpression of highly homologous genes tends to result genetic instability (i.e. undesirable homologous recombination). Expressing crtW genes having relatively low to moderate sequence homology should decrease the probability of genetic instability normally associated with expression of highly homologous genes. This is particularly important when developing genetically-stable commercial strains for optimal production of the desired product (i.e. ketocarotenoids).
CrtW genes having divergent nucleotide sequences are most suitable for expressing multiple ketolases in a single recombinant host cell. This is especially important when ketolase activity becomes the rate-limiting step in the ketocarotenoid biosynthesis pathway. Increasing the number of crtW genes that can be simultaneously expressed in the production host is expected to increase ketocarotenoid production, assuming that the pool of available substrates is not limiting.
Additionally, CrtW ketolases tend to exhibit substrate flexibility. However, it can be envisioned that different CrtW ketolases may exhibit preferential activity towards one or more possible substrates (i.e. β-carotene versus zeaxanthin). Simultaneous expression of multiple CrtW ketolases, each selected based on their preferred substrate, may be used for optimal production of a desired ketocarotenoid. One of skill in the art may optimize production of the desired ketocarotenoid end product by analyzing the available substrate pool within the desired host cell, selectively expressing an appropriate combination of ketolases for optimal production of the desired ketocarotenoid.
The problem to be solved therefore is to identify and isolate novel crtW ketolase genes useful for engineering production of ketocarotenoids (i.e. canthaxanthin and astaxanthin). The present invention has solved the stated problem by providing three novel crtW genes useful for the production of ketocarotenoids in recombinant host cells. Methods for producing ketocarotenoids using the present CrtW ketolases are also provided.